Impact of Envelope Cholesterol and Spike gp41 on Cell-independent Lytic Inactivation of HIV-1 by Peptide Triazole Thiols

HIV-1 is a retrovirus that infects host cells carrying the receptor CD4 and the co-receptor CCR5/CXCR4. The process of infection is carried out by the virus specific proteins gp120 and gp41, expressed as a trimer of dimers on the virus surface. This interaction can be interrupted with the use of peptide triazole thiols (PTT). They are a family of entry inhibitors that carry dual antagonist behavior against gp120 by blocking both CD4 and co-receptor interactions. The thiol introduced into the PTT sequence by a C-terminal Cysteine adds an additional irreversible inactivating step consisting of lysis leading to the release of capsid p24 protein from the lumen of the virus. Since PTTs do not interact with the membrane as established with viral particles with no spike or particles pseudotyped with VSV-G, lysis must be a consequence of conformational changes within the spike being triggered by PTTs, resulting in membrane perturbation and the eventual mixing of viral luminal contents with the extracellular surroundings. Since there is a similar mixing of viral contents with intracellular contents after CD4/co-receptor interaction with the virus, we decided to use this lytic event as a window to study the lipid-protein interactions that take place to allow the disruption of the membrane and the eventual release of luminal contents. This study was split into two sections. In the first section, the lipids that make up the viral lipid bilayer (envelope) were investigated and a thorough survey of literature pointed to cholesterol, the major lipid constituent (ca. 45 mol %). Prior literature has shown that depletion with a chemical agent specific for cholesterol, methyl beta-cyclodextrin (MβCD) from the viral envelope or from cells producing viruses resulted in a complete loss of infectivity. When tested for the impact of sterol depletion on lysis with PTT, the results were dramatic. Small amounts of MβCD treatment ( 312 μM MβCD) using the probe Laurdan and a bell-shaped quenching of fluorescence using the probe Dehydroergosterol with a nadir in intensity at 312 μM MβCD. These data suggested that the membrane was undergoing morphological changes with the depletion of cholesterol and this was affecting the lysis observed with PTTs and infectivity. The sterol data and DHE data hint at the role of rafts in the transitions observed but this has not been conclusively proven. To further the understanding, the protein involved with the membrane gp41 was investigated. Different regions of gp41 were examined for their role in lysis through site-directed mutagenesis of the BaL.01 sequence. Of the mutants created, all showed dose-dependent lytic release with PTT treatment in comparable levels and IC50s to the wild-type BaL.01 pseudovirus. While all mutants showed reduced infectivity, which was consistent with literature, mutations at the putative interface between envelope cholesterol and the spike (CRAC -> L676I, C-terminal tail truncation -> R706St) showed enhancement of lysis at [MβCD] lower than that for wild type (~ 10 μM). One possible reason might be that envelope cholesterol that is held tightly by viral spike interactions is more easily removed by MβCD in the mutants. Mutations that targeted conserved tryptophan residues within the membrane proximal external region (MPER domain) affected the sensitivity to cholesterol depletion, with the mutant containing all Trp residues mutated (W(1-5)A) being the least sensitive. Since these residues are known to be critical for infectivity of HIV-1 and other viruses including Influenza and Ebola, the data suggest a common purpose for this region in both infectivity and lysis. Based on the mutational data collected, one may conclude the following: (1) Mutations have a much bigger effect on infectivity than on lysis. (2) Multiple regions of gp41 might be involved in the lytic mechanism, and mutations targeting single regions might not be big enough to stop lysis. (3) Alternatively, none of the regions targeted with mutations are crucial for lysis, though this is very unlikely due to the trends observed with sensitivity to cholesterol. Taken in context with the cholesterol depletion data, the findings can be explained with an energy to reaction argument. High cholesterol content (~ 45 mol %) results in low fluidity and tight packing of phospholipids, and this might benefit the spike in helping it maintain structure and conformation. However, it raises the energy barrier for the membrane-interacting gp41 protein in processes such as fusion and lysis which require large conformational changes such as the formation of the 6-helix bundle. Removing cholesterol to a limited extent might help lower the barrier, permitting these events to occur at a higher frequency and greater likelihood and this may be why we see an enhancement in lysis and infection. Investigations into the lytic mechanism with PTTs have provided a potential window into the mechanism of fusion that occurs with HIV-1. This is of critical importance, as there is a pressing need for entry inhibitors and a better understanding of the mechanism might foster a whole new generation of virus-inactivating, lytic entry inhibitors.Ph.D., Biomedical Engineering -- Drexel University, 201

Vesicle capture by membrane-bound Munc13-1 requires self-assembly into discrete clusters

Munc13-1 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13-1 form nano-assemblies in active zones of neurons. However, it is not known whether such clustering of Munc13-1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13-1 clustering is also unknown. Here, we report that nano-clustering is an inherent property of Munc13-1 and is indeed required for vesicle binding to bilayers containing Munc13-1. Purified Munc13-1 protein reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ˜ 20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters containing a minimum of 6 copies of Munc13-1 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13-1 is not required for Munc13-1 clustering, but is required for efficient vesicle capture. This capture is largely due to a combination of electrostatic and hydrophobic interactions between the C2C domain and the vesicle membrane

Macrocyclic Envelope Glycoprotein Antagonists that Irreversibly Inactivate HIV‑1 <i>before</i> Host Cell Encounter

We derived macrocyclic HIV-1 antagonists as a new class of peptidomimetic drug leads. Cyclic peptide triazoles (cPTs) retained the gp120 inhibitory and virus-inactivating signature of parent PTs, arguing that cyclization locked an active conformation. The six-residue cPT <b>9</b> (AAR029b) exhibited submicromolar antiviral potencies in inhibiting cell infection and triggering gp120 shedding that causes irreversible virion inactivation. Importantly, cPTs were stable to trypsin and chymotrypsin compared to substantial susceptibility of corresponding linear PTs

Peptide Triazole Inactivators of HIV‑1 Utilize a Conserved Two-Cavity Binding Site at the Junction of the Inner and Outer Domains of Env gp120

We used coordinated mutagenesis, synthetic design, and flexible docking to investigate the structural mechanism of Env gp120 encounter by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated that the PT class of inhibitors suppresses binding at both CD4 and coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent irreversible virus inactivation. Despite these enticing anti-HIV-1 phenotypes, structural understanding of the PT–gp120 binding mechanism has been incomplete. Here we found that PT engages two inhibitor ring moieties at the junction between the inner and outer domains of the gp120 protein. The results demonstrate how combined occupancy of two gp120 cavities can coordinately suppress both receptor and coreceptor binding and conformationally entrap the protein in a destabilized state. The two-cavity model has common features with small molecule gp120 inhibitor binding sites and provides a guide for further design of peptidomimetic HIV-1 inactivators based on the PT pharmacophore

Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV‑1 by Peptide Triazole Thiols

We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine–ferrocenyltriazole-proline–tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)­phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol–disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection